Use of aspartic proteases in cosmetics and therapeutics

ABSTRACT

The invention provides a cosmetic or pharmaceutical composition comprising, in a physiologically acceptable medium, at least one purified natural or synthetic polypeptide wherein the peptide sequence is represented wholly or partly by at least one sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 16, SEQ ID NO: 25 and SEQ ID NO: 27 and their homologs.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the US. national stage of International ApplicationNo. PCT/FR03/002151, filed Jul. 9, 2003, and claims priority under 35U.S.C. §119 of French Patent Application No. 02/08613, filed Jul. 9,2002, said applications being incorporated by reference herein in theirentireties and relied upon.

The present invention relates mainly to the use, in cosmetics andtherapeutics, of a novel aspartic acid protease, referred to as SASPase,of the truncated or derived forms of said protein or of a mixture ofpolypeptides derived from the proteolysis thereof, in particular for thepurpose of treating conditions related to a dysfunction of cellproliferation and/or differentiation.

The invention also relates to deoxyribonucleic acid sequences encodingsaid aspartic acid protease SASPase and its “activated” forms, to thecorresponding polypeptide sequences and to the uses of saiddeoxyribonucleic acid sequences.

Proteases are hydrolytic enzymes capable of cleaving peptide bonds. Acertain number of them are, today, known to play an essential role inthe equilibrium of the physiology of the epidermis.

The epidermis is conventionally divided into a basal layer ofkeratinocytes constituting the germinative layer of the epidermis, a“spiny” layer consisting of several layers of polyhedral cells arrangedon the germinative layers, one to three “granular” layers consisting offlattened cells containing distinct cytoplasmic inclusions, thekeratohyalin granules, and finally, a set of upper layers, calledcornified layers (or stratum corneum), consisting of keratinocytes atthe terminal stage of their differentiation, called corneocytes.

Corneocytes are anucleated cells consisting mainly of a fibrous materialcontaining cytokeratins, surrounded by a cornified envelope. There is apermanent production of new keratinocytes in order to compensate for thecontinuous loss of epidermal cells in the cornified layer according to amechanism called desquamation. An imbalance between the production ofcells in the basal layer and the rate of desquamation can in particularresult in the formation of scales at the surface of the skin.

As it happens, many skin pathologies are characterized by the productionof a thick cornified layer and by abnormal desquamation, i.e.hyperkeratosis. By way of example, mention may be made of:

-   -   xerosis (or dryness of the skin),    -   ichthyoses,    -   psoriasis,    -   certain benign or malignant tumor lesions, and    -   reactive hyperkeratoses.

Conversely, certain pathological manifestations result in a thinning ofthe epidermis, and more particularly of the cornified layer. This typeof manifestation then results in excessive fragility of the cutaneouscovering. By way of representation of these conditions, mention may inparticular be made of reactions of immune origin, generally induced bybeing in the presence of or coming into contact with one or moreexogenous agents.

Consequently, knowledge of the polypeptides involved in intercorneocytecohesion is one of the pathways which may make it possible to developproducts intended to combat the effects of an excess of or a deficiencyin one or more polypeptide(s) of this type, in particular at the surfaceof the skin.

One of the objects of the invention is precisely to propose the use, forcosmetic and/or therapeutic purposes, of a polypeptide involved inregulating the phenomenon of epidermal differentiation/proliferation.

More precisely, the inventors have demonstrated in human keratinocytes,isolated and purified a polypeptide having, in its peptide sequence (SEQID NO 5), the sequence FLVDSGAQVSVV (SEQ ID NO: 1) corresponding to aPROSITE signature PS00141 of active sites of proteases of the “asparticacid” protease family.

This polypeptide, also referred to hereinafter as SASPase protein, is,moreover, characterized by the presence, in its peptide sequence, of thefollowing sequences:

AQFLVANASAEEAIIGTDVLQ (SEQ ID NO: 2) and ILGVWDTAV (SEQ ID NO: 3).

Unexpectedly, the inventors have demonstrated that the proteinrepresented by the sequence SEQ ID NO: 5, also referred to as SASPaseprotein, have significant proteolytic activity, and have in particularnoted this activity with respect to casein and to insulin, as shown inthe examples hereinafter.

They have, moreover, observed that this SASPase protein is autocatalyticand generates, at a pH of between 3 and 7, and preferably greater thanor equal to 4.5, a truncated form referred to as “activated SASPase”,corresponding to the sequence SEQ ID NO: 6 capable, in turn, ofdimerizing.

One aspect of the invention therefore relates to an isolated andpurified polypeptide belonging to the aspartic acid protease family,characterized in that it has a peptide sequence represented by thesequence SEQ ID NO: 6, SEQ ID NO: 16, SEQ ID NO: 25 or SEQ ID NO: 27.

SEQ ID NO: 6 corresponds to an activated form of SEQ ID NO: 5.

SEQ ID NO: 16 corresponds to the sequence SEQ ID NO: 6 which has had itsfirst two amino acids deleted.

The sequence SEQ ID NO: 25 is another activated form of SEQ ID NO: 5. Itis obtained from a truncated form of SASPase (SEQ ID NO: 36) from whichthe site encoding the sequence FANS (SEQ ID NO: 29) has been deleted. Itis more particularly generated at pH 5.00 in acetate buffer.

The sequence SEQ ID NO: 27 corresponds to the sequence SEQ ID NO: 25from which a part of its C-terminal fragment has been deleted.

In general, the invention extends to all the homologous forms of thevarious polypeptides or peptide sequences mentioned. Conventionally, theexpression “homolog of a polypeptide or of a peptide sequence” isintended to mean any polypeptide or any peptide sequence having at least85%, especially at least 90%, and in particular at least 95% sequencehomology, and having, where appropriate, the same type of biologicalactivity as said polypeptide or said peptide sequence.

These homologous forms encompass the variants defined hereinafter.

The invention extends in particular to the homologous forms of theabovementioned polypeptides, i.e. the forms showing the same biologicalactivity and having at least 85%, especially at least 90%, and inparticular at least 95%, sequence homology with the sequence SEQ ID NO:6, SEQ ID NO: 16, SEQ ID NO: 25 or SEQ ID NO: 27.

Similarly, the invention extends to the proteins having at least 30%homology with the sequence SEQ ID NO: 6, SEQ ID NO: 16 or SEQ ID NO: 25,on the condition that the homology with the sequence of the active siteSEQ ID NO: 1 contained in SEQ ID NO: 6, SEQ ID NO: 16, SEQ ID NO: 25 andSEQ ID NO: 27 is at least 80%.

The invention also extends to the proteins having both the “aspartylprotease retroviral type” unit defined under the PROSITE unit reference:PS50175, and also at least one transmembrane domain as predicted by thealgorithms recognized for such a detection, among which mention may bemade of: PRED-TMR2, TMHMM, TMpred and SOSUI.

The modifications, also referred to as mutations or variations,according to the invention can derive either from the deletion of one ormore amino acids of the sequence SEQ ID NO: 6, SEQ ID NO: 16, SEQ ID NO:25 or SEQ ID NO: 27, or from the addition of one or more amino acids tothe sequence SEQ ID NO: 6, SEQ ID NO: 16, SEQ ID NO: 25 or SEQ ID NO:27, or alternatively from the substitution of one or more amino acidsfor the sequence SEQ ID NO: 6, SEQ ID NO: 16, SEQ ID NO: 25 or SEQ IDNO: 27. The corresponding sequences are also referred to under the term“variants” in the context of the present invention.

By way of illustration of the deletion variants, mention may moreparticularly be made of the following peptide sequences:

the sequence SEQ ID NO: 4 corresponding to SEQ ID NO: 5 truncated interms of its N-terminal fragment Δ1-84;

the sequence SEQ ID NO: 7 corresponding to the sequence of theN-terminal portion of the SASPase protein (SEQ ID NO: 5). Theinteraction of this fragment with a biological ligand could be animportant step for the activation of the SASPase protein, and inparticular the generation of its activated form (SEQ ID NO: 6);

the sequence SEQ ID NO: 8 corresponding to the sequence of the“transmembrane” portion of the SASPase protein (SEQ ID NO: 5);

the sequence SEQ ID NO: 9 corresponding to the sequence of a peptidederived from the C-terminal portion of the SASPase protein (SEQ ID NO:5);

the sequences SEQ ID NO: 29 and SEQ ID NO: 30 corresponding to twoactivation sites.

Also covered under the term “variant” are the proteins from fusion ofthe SASPase protein (SEQ ID NO: 5), or of its activated forms SEQ ID NO:6, SEQ ID NO: 16, SEQ ID NO: 25 or SEQ ID NO: 27, with anotherpolypeptide, a hydrophilic or hydrophobic targeting agent or abioconversion precursor capable in particular of controlling theactivation of said protein.

It is known that polypeptides can undergo post-translationalmodifications such as the formation of disulfide bonds, specificproteolytic cleavages, the addition of carbohydrates (glycosylation),phosphorylation, in particular on serines and/or threonines and/ortyrosines, and/or association with lipids.

The polypeptide of the invention may have undergone one or morepost-translational modifications. In particular, the polypeptidesaccording to the invention can be N-glycosylated, phosphorylated,myristoylated, amidated and/or citrilinated.

Thus, the invention also relates to the claimed polypeptides which mayor may not have undergone post-translational modifications.

The invention also extends to the multimeric forms, and preferably tothe dimeric form of the peptide sequence SEQ ID NO: 6, SEQ ID NO: 16,SEQ ID NO: 25 or SEQ ID NO: 27. The dimeric form of the peptide sequenceSEQ ID NO: 6 is in particular characterized in example V hereinafter. Itcan in particular be obtained by molecular association of the monomericform or by expression of its cDNA encoding the active dimer,incorporating, by way of bonding agent between the two monomericentities, a unit made up of 2 to 6 amino acids.

The claimed polypeptides may be of natural or synthetic origin. The term“synthetic” is here intended to mean any polypeptide obtained chemicallyor by production in an organism after introduction into this organism ofthe elements required for this production.

They may be derived from any possible origin, namely either animal, inparticular mammalian or even more particularly human origin, or plantorigin, or from microorganisms (for example viruses, phages, bacteria,yeast, inter alia) or else from fungi, or derived from overexpression ina eukaryotic system, for example a mammalian cell, without any prejudiceas to whether or not they are naturally present in said organism oforigin.

In particular, the polypeptides in accordance with the invention are ofnatural origin, purified from mammalian tissues, more particularly frommammalian skin.

In particular, they are purified from human skin, and even moreparticularly from human epidermis.

It is known that, in a polypeptide, one or more amino acid residues canbe replaced with amino acid residues having a similar hydropathic indexwithout, however, changing the biological properties of the polypeptide.

The hydropathic index is an index assigned to amino acids as a functionof their hydrophobicity and of their charge (Kyte et al. (1982), J. Mol.Biol., 157: 105).

Thus, a subject of the invention is also a polypeptide as describedabove, in which at least one amino acid residue has been replaced withan amino acid residue having a similar hydropathic index.

The polypeptides can also be classified according to their isoelectricpoint.

The theoretical isoelectric point of a polypeptide can be deduced fromits amino acid chain. The polypeptides of the invention aretheoretically acid polypeptides.

Thus, the polypeptides of sequence SEQ ID NO: 5, SEQ ID NO: 6 or SEQ IDNO: 16 have an isoelectric point of between 3 and 9, more particularlyof between 4 and 6, and especially of approximately 5.8.

It is also known that the primary amino acid sequence and also thevarious post-translational modifications that a polypeptide undergoesmean that said polypeptide can be characterized by its apparentmolecular mass expressed in kilodaltons.

The term “apparent molecular mass” is intended to mean the molecularmass obtained for the polypeptide by comparison of the electrophoreticmobility thereof with those of standard proteins of known molecularweights on polyacrylamide/sodium dodecyl sulfate gel, or else bycomparison of the polypeptide elution volume with that of standardproteins of known molecular weights in exclusion chromatography(according to the techniques described in “Protein Purification”, J-C.Janson and L. Ryden, VCH Publisher Inc. N.Y., 1989). (The methodselected in the context of the invention is that based onelectrophoretic mobility).

Knowledge of the amino acid chain of the polypeptide of the inventionmakes it possible to determine the theoretical molecular weight thereof.

The invention therefore relates to a polypeptide of sequence SEQ ID NO:6 having an apparent molecular mass of between 5 and 30 kilodaltons(kD), especially of between 9 and 15 kD, and more particularly ofbetween 11 and 14 kD. In particular, this polypeptide of the inventionhas an apparent molecular mass of the order of 12 kD.

As emerges from the examples presented hereinafter, the inventors havecharacterized the expression of the polypeptide for which the sequenceis represented by the sequence SEQ ID NO: 5, in a large number of humanbiological tissues such as fetal liver, placenta, muscle, lung or smallintestine, and especially in the brain and the heart, and moreparticularly in the epidermis where the expression is particularly high.

It has, moreover, been noted that the SASPase of sequence SEQ ID NO: 5degrades corneodesmosin, which is a marker of desquamation.

Finally, the presence, in the polypeptide sequence, of the SASPase (SEQID NO: 5), of an autocatalytic site corresponding to a site describedfor protease of the matrilysin type capable of activating type 1, 2 and9 MMPs, reflects a potential activity of the SASPase, and also of itsactivated forms (SEQ ID NO: 6, SEQ ID NO: 16, SEQ ID NO: 25 or SEQ ID NO27) or of its fragments, on endogenous substrates, and therefore ofpotential applications in cicatrization, re-epithialization, aging,angiogenesis and cancerization (invasion process) for said protein andits various activated forms or fragments.

All this information therefore validates the involvement of thepolypeptide in accordance with the invention in the process of cellproliferation and/or differentiation and identifies it as a noveldermato/cosmetological and therapeutic target.

Consequently, a second aspect of the invention relates to a cosmetic orpharmaceutical composition comprising, in a physiologically acceptablemedium, at least one purified, natural or synthetic polypeptide, thesequence of which comprises at least one peptide sequence representedwholly or partly by at least one sequence chosen from SEQ ID NO: 1, SEQID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 9, SEQ ID NO: 16, SEQ ID NO: 25 and SEQ ID NO: 27 and homologsthereof.

In particular, the present invention relates to a cosmetic orpharmaceutical composition comprising, in a physiologically acceptablemedium, at least one purified, natural or synthetic polypeptide, thepeptide sequence of which is represented wholly or partly by thesequence SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 16 or SEQID NO: 25 or SEQ ID NO: 27, or homologs thereof, and in particular whichis represented by the peptide sequence SEQ ID NO: 5, SEQ ID NO: 6, SEQID NO: 7, SEQ ID NO: 16, SEQ ID NO: 25 or SEQ ID NO: 27.

The present invention also relates to a cosmetic or pharmaceuticalcomposition comprising at least one purified, natural or syntheticpolypeptide, the peptide sequence of which is represented wholly orpartly by at least one sequence chosen from SEQ ID NO: 1, SEQ ID NO: 4,SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,SEQ ID NO: 16, SEQ ID NO: 25 and SEQ ID NO: 27, and homologs thereof,and in particular at least one polypeptide of sequence SEQ ID NO: 5, SEQID NO: 6, SEQ ID NO: 7, SEQ ID NO: 16, SEQ ID NO: 25 or SEQ ID NO: 27,in a multimeric, and preferably dimeric, form.

For the purpose of the invention, and unless otherwise indicated, theterm “polypeptide” is intended to cover, in the claimed compositions,natural or synthetic polypeptides, whether they are obtained byproteolysis or by synthesis, the various post-translational formsthereof and in particular those described above, or else any natural orsynthetic polypeptide, the sequence of which consists wholly or partlyof the abovementioned sequences, such as, for example, the variantsdescribed above.

The present invention also relates to a cosmetic or pharmaceuticalcomposition in which said polypeptide is in the form of a polypeptide ofsequence SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 16, SEQ IDNO: 25 or SEQ ID NO: 27, fused with another polypeptide, a hydrophilicor hydrophobic targeting agent or a bioconversion precursor.

It is, moreover, known that the primary amino acid sequence of apolypeptide determines sites specifically recognized by proteases which,once the recognition of these sites is effective, will, with or withoutbinding to said polypeptide, induce cleavage thereof by proteolysis.

Consequently, the invention is also directed toward a cosmetic orpharmaceutical composition comprising, in a physiologically acceptablemedium, at least one polypeptide mixture derived from the proteolysis ofa polypeptide, the sequence of which is represented wholly or partly bythe sequence SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 16,SEQ ID NO: 25 or SEQ ID NO: 27, or homologs thereof, and moreparticularly the sequence of which is represented by SEQ ID NO: 5, SEQID NO: 6, SEQ ID NO: 7, SEQ ID NO: 16, SEQ ID NO: 25 or SEQ ID NO: 27.

The amount of polypeptide contained in the compositions of the inventiondepends, of course, on the desired effect and can therefore vary to alarge extent.

To give an order of magnitude, the composition can contain a polypeptidein accordance with the invention in an amount representing from 0.00001%to 50% of the total weight of the composition, and preferably in anamount representing from 0.001% to 10% of the total weight of thecomposition, and even more preferably in an amount representing from0.1% to 1% of the total weight of the composition.

As described above, a certain number of disorders are associated withcell differentiation and/or proliferation conditions. Insofar as thepolypeptides in accordance with the invention are involved in theregulation of these two phenomena, they advantageously constitutepotential targets for treating any disorder resulting from a dysfunctionof cell proliferation or differentiation, in particular epidermal cellproliferation or differentiation. Consequently, besides the fact thatthe polypeptides according to the invention can be used directly asactive material in a cosmetic or pharmaceutical composition, they canalso themselves serve as a target in a cosmetic or pharmaceuticaltreatment or be used as diagnostic tools.

The inventors have, moreover, characterized the presence of cleavagesites in the peptide sequence SEQ ID NO: 5, partly located in itsN-terminal portion and also present in the active forms SEQ ID NO: 6 andSEQ ID NO: 16 and partly present in its C-terminal portion and alsopresent in the active forms SEQ ID NO: 25 and SEQ ID NO: 27. Moreprecisely, the deduced cleavage sites for the autoactivation of theSASPase itself are F/A, N/S, E/L, A/L, R/F, H/S, F/E, E/A. Evidently,these cleavage sites may be determinant in the activation of theprotein, or for blocking it, or conversely for activating itshydrolysis.

The relevant sites more precisely located in the N-terminal region areF/A and N/S, and appear in the FANS sequence referred to as SEQ ID NO:29. They appear in particular to be involved in generating the activatedform represented by SEQ ID NO: 25.

As regards the second potential region for activation or inactivation ofthe SASPase protein SEQ ID NO: 5, it corresponds more particularly tothe sequence DLELIE, referred to as SEQ ID NO: 30, and comprises inparticular the cleavage site E/L.

Based on these cleavage sites, it is possible to envision synthesizingvarious types of peptides that are either modified or carry a quenchedfluorophor, and which will serve as a substrate or as inhibitors.

The minimum sequence that can be used is obviously the dipeptide (aminoacids on either side of the cleavage site); however, in general,peptides of 8 to 12 amino acids where the cleavage site is in thecentral position are used. For example, it is known that the SASPasecleaves insulin at the E/A position. It is therefore possible to imaginedeveloping a peptide-type substrate that incorporates this cleavagesite, modifying it chemically at each of its ends with a quenchedfluorophor group of the type: Abz(NO₂)Tyr (N-end: aminobenzoic acid:C-end: nitrotyrosine (amide)). Hydrolysis of this peptide with theprotease will separate the fluorophor and the quencher and willtherefore be followed by an increase in fluorescence.

Another chemical couple such as Dabcyl/EDANS can be used to createanother type of quenched substrate.

It is also possible to envision a chromogenic substrate by coupling thepeptide with the para-nitroanilide group.

In the same way, it is possible to envision developing anSASPase-specific inhibitor by replacing one of the amino acids of such apeptide, by modifying the peptide bond or by adding a chemical group sothat the bond is made non-hydrolyzable by the enzyme but the peptidestill has affinity for the active site. For example, a non-natural aminoacid is added, the peptide bond is reduced, or chemical groups of thealdehyde, chloromethyl ketone or diazomethyl ketone type are added.

The inventors have in particular shown that point modificationsintroduced into the peptide substrate represented by SEQ ID NO: 30 mayhave a significant effect on their affinity for the enzyme.

More precisely, the corresponding modified sequences prove to be capableof constituting potential SASPase inhibitors or activators.

By way of illustration of these potential modulators, peptide substrateshaving at least the following sequences may more particularly beproposed:

EFDLELIEED SEQ ID NO: 31 EFDLDLIEED SEQ ID NO: 32 EFDLDLIEWD SEQ ID NO:33 EFDLDLIHWD SEQ ID NO: 34 EPNLDLIEED SEQ ID NO: 35

An activator effect of the substrates represented by the sequences SEQID NO: 31 and SEQ ID NO: 32 has in particular been noted.

Consequently, the present invention also relates to the use of achemical or biological compound, for preparing a composition intended tointeract with a polypeptide, the peptide sequence of which comprises atleast one sequence chosen from SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5,SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 16,SEQ ID NO: 25, SEQ ID NO: 27 and SEQ ID NO: 30, and homologs thereof,and more particularly with a polypeptide of sequence SEQ ID NO: 5, SEQID NO: 6, SEQ ID NO: 7, SEQ ID NO: 16, SEQ ID NO: 25, SEQ ID NO: 27 orSEQ ID NO: 30, or to modulate the biological activity thereof.

This biological compound may in particular be a protease having aspecific site for recognition and/or for binding and for cleavage withinthe amino acid sequence of said polypeptide, and preferably of apolypeptide having, as primary sequence, the sequence SEQ ID NO: 5, SEQID NO: 6, SEQ ID NO: 7, SEQ ID NO: 16, SEQ ID NO: 25 or SEQ ID NO: 27.

As it happens, it has been shown that inhibitors of proteases of theretropepsin type also show an effect with respect to the activity of theSASPase protein. By way of illustration of the inhibitors which may beused according to the invention, mention may in particular be made ofretropepsin inhibitors, in particular sold by Bachem.

This inhibitor may also be selected so as to interfere with thedimerization of the SASPase (SEQ ID NO: 5) or of one of its activatedforms represented by SEQ ID NO: 6, SEQ ID NO: 16, SEQ ID NO: 25 or SEQID NO: 27, prior to its proteolytic activity. This inhibitor may also bean endogenous inhibitor capable of specifically inhibiting the SASPaseor its autoactivation.

Similarly, this biological compound may be an activator. By way ofrepresentation thereof, mention may in particular be made of the RP3retropepsin modulator characterized in example VIII hereinafter.

It may also be an antibody specific for said polypeptide.

Similarly, the present invention extends to the use of a biological orchemical compound, for preparing a composition intended to inhibit thedimerization of the polypeptide of sequence SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 16, SEQ ID NO: 25 or SEQ ID NO: 27.

A subject of the present invention is also the use of a polypeptide, thesequence of which comprises at least, and in particular is representedby, a sequence chosen from SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33,SEQ ID NO: 34 and SEQ ID NO: 35, for preparing a composition intended tomodulate the activity of the SASPase.

The pharmaceutical or cosmetic compositions claimed and consideredaccording to the invention may be compositions used in the cosmetics,dermatological, dermatocosmetics and pharmacological fields.

A physiologically acceptable medium is, according to the invention, acosmetically or pharmaceutically acceptable medium that is compatiblewith the skin, the mucous membranes, and nails and/or the hair.

The compositions according to the invention can be applied to the nails,the hair, and more particularly to the skin and the mucous membranes.

They are particularly advantageous for acting on one or more epidermalmechanisms such as the degradation of protein(s), the activation ofenzyme(s), and/or the regulation of the epidermaldifferentiation/proliferation phenomenon.

As it happens, the compositions according to the invention areparticularly useful for compensating for an imbalance in epidermaldifferentiation/proliferation. More particularly, they may be useful forregulating the phenomena of moisturization, of inflammation, ofmelanogenesis and/or of desquamation, the aging phenomenon, the defensemechanisms, for the regulation of differentiation/proliferation oncertain cell and skin types: keratinocytes, melanocytes, Langerhanscells, sebocytes, adipocytes, and also the regulation of secretionphenomena and of invasion processes.

More precisely, the claimed compositions prove to be advantageous in thefollowing areas:

-   -   for treating dermatological complaints associated with a        keratinization disorder relating to differentiation and to        proliferation, in particular for treating common acne,        comedo-type acne, polymorphic acne, rosacea, nodulocystic acne,        acne conglobata, senile acne, and secondary acne such as solar,        drug-related or occupational acne,    -   for treating other types of keratinization conditions, in        particular ichtyoses, ichtyosiform conditions, Darrier's        disease, palmoplantar kerato-derma, leukoplakia and        leukoplakiform conditions, and cutaneous or mucosal (oral)        lichen,    -   for treating other dermatological complaints associated with a        keratinization condition, with an inflammatory and/or        immunoallergic component, and in particular all forms of        psoriasis, whether cutaneous, mucosal or ungual psoriasis, and        even psoriatic rheumatism, or alternatively cutaneous atopy,        such as eczema, or urticaria or else gingival hypertrophy; the        compounds may also be used in certain inflammatory ailments not        exhibiting any keratinization condition,    -   for treating all dermal or epidermal proliferations, whether        benign or malignant, whether or not of viral origin, such as        common warts, flat warts and epidermodysplasia verruciformis,        oral or florid papillomatoses, and proliferations which may be        induced by ultraviolet light, in particular in the case of        basal-cell and spinocellular epithelioma,    -   for treating other dermatological disorders such as bullous        dermatoses and collagen diseases,    -   for repairing or combating skin aging, whether it is        photoinduced or chronological, or for reducing actinic keratoses        and pigmentations, or any pathological conditions associated        with chronological or actinic aging,    -   for preventing or curing stigmata of epidermal and/or dermal        atrophy induced by local or systematic corticosteroids, or any        other form of skin atrophy,    -   for preventing or treating cicatrization conditions or for        preventing or repairing stretch marks, and    -   for combating sebaceous function conditions such as        hyperseborrhea of acne or simple seborrhea.

In the case of an application in the cosmetic field, in particular forbody and hair hygiene, the compositions according to the invention arein particular useful for treating skin with a tendency to suffer fromacne, for hair regrowth, for anti-hairloss, for combating the greasyappearance of the skin or of the hair, in protection against the harmfulaspects of the sun or in the treatment of physiologically dry skin, forpreventing and/or combating photoinduced or chronological aging. Theymay also be useful for improving reconstructed skin. The polypeptideand/or its derivatives and/or modulators of its activity or of itsactivation can also be used directly in the culture medium.

Another subject of the invention is a method of cosmetic treatmentintended to combat skin conditions associated with a dysfunction of cellproliferation and/or differentiation, such as in particular dry skin,hyperkeratosis, parakeratosis, sebogenesis conditions, neoplasias and/orsigns of skin aging, characterized in that a cosmetic compositioncomprising at least one polypeptide, the peptide sequence of whichcomprises at least one sequence chosen from SEQ ID NO: 1, SEQ ID NO: 4,SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,SEQ ID NO: 16, SEQ ID NO: 25 and SEQ ID NO: 27, and in particular whichis represented wholly or partly by SEQ ID NO: 5, SEQ ID NO: 6, SEQ IDNO: 7, SEQ ID NO: 16, SEQ ID NO: 25 or SEQ ID NO: 27, and moreparticularly which has the sequence SEQ ID NO: 5, SEQ ID NO: 6, SEQ IDNO: 7, SEQ ID NO: 16, SEQ ID NO: 25 or SEQ ID NO: 27, or a mixturederived from the proteolysis of one of these polypeptides, is applied tothe skin, the mucous membranes and/or the keratin fibers.

The method of treatment of the invention is a cosmetic method intendedto improve the esthetic appearance of the individual experiencingepidermal proliferation and/or differentiation conditions.

The invention also relates to the use of a polypeptide, the peptidesequence of which comprises at least one sequence chosen from SEQ ID NO:1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8,SEQ ID NO: 9, SEQ ID NO: 16, SEQ ID NO: 25 and SEQ ID NO: 27, and inparticular which is represented wholly or partly by SEQ ID NO: 5, SEQ IDNO: 6, SEQ ID NO: 7, SEQ ID NO: 16, SEQ ID NO: 25 or SEQ ID NO: 27, andmore particularly which has the sequence SEQ ID NO: 5, SEQ ID NO: 6, SEQID NO: 7, SEQ ID NO: 16, SEQ ID NO: 25 or SEQ ID NO: 27, or of a mixturederived from the proteolysis of one of these polypeptides, for preparinga pharmaceutical composition intended for the treatment ofdermatological ailments, and in particular those mentioned above.

In particular, the invention relates to the use of a polypeptide or of amixture as described above, for preparing a pharmaceutical compositionintended to treat ichtyosis, psoriasis or any pathology involvinghyperkeratosis or parakeratosis or having an inflammatory component.They may also be pain-killing compositions, or compositions for treatingcertain skin diseases such as eczema, rosacea, psoriasis, lichens orsevere pruritus.

Insofar as the polypeptides of sequence SEQ ID NO: 5, SEQ ID NO: 6, SEQID NO: 16, SEQ ID NO: 25 or SEQ ID NO: 27 have considerable structuralhomologies with retroviral proteins, as is shown in the examples and inFIG. 4, an in particular with those of the human immunodeficiency virus,they are also capable of behaving like agents capable of modulatingviral infection or of being modulated by certain viral antiproteases.

Consequently, a subject of the invention is also the use of apolypeptide, the peptide sequence of which comprises at least onesequence chosen from SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ IDNO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 16, SEQ IDNO: 25 and SEQ ID NO: 27, and homologs thereof, and in particular isrepresented wholly or partly by SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO:7, SEQ ID NO: 16, SEQ ID NO: 25 and SEQ ID NO: 27, and more particularlywhich has the sequence SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO: 16, SEQ ID NO: 25 and SEQ ID NO: 27, for preparing an antiviralcomposition.

These compositions may in particular be useful for treating epidermalpathologies associated with a virus of the papillomavirus, Herpes or HIVtype.

Similarly, such compositions may be advantageous for treating sideeffects related to inhibition of the endogenous SASPase by the medicinalproducts directed against these viruses or other pathological viruses.More particularly, this specific application of the polypeptidesaccording to the invention takes advantage of the structural homologyobserved between the SASPase and viral retropepsins, as illustrated bythe examples hereinafter.

The treatment generally involves application of the composition asdescribed above to the skin of the individual to be treated.

A subject of the present invention is also the use of a polypeptide, thepeptide sequence of which is chosen from the sequences SEQ ID NO: 1, SEQID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO 9, SEQ ID NO: 16 and SEQ ID NO: 25 or SEQ ID NO 27, as a tool in adiagnostic or screening method, or for preparing a diagnostic tool.

More precisely, the invention is directed toward the use of apolypeptide, the peptide sequence of which is chosen from SEQ ID NO: 1,SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8,SEQ ID NO 9, SEQ ID NO: 16, SEQ ID NO: 25 and SEQ ID NO: 27, andhomologs thereof, more particularly of a polypeptide which has thesequence SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 16, SEQ IDNO: 25 or SEQ ID NO: 27, or of its proteolytic fragments and of anysynthetic peptide deduced from its sequence, for preparing or purifying,optionally from epidermis, any molecule capable of modulating itsinteraction with possible ligands.

In addition, the invention is directed toward the use of a polypeptide,the peptide sequence of which is chosen from SEQ ID NO: 1, SEQ ID NO: 4,SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,SEQ ID NO: 16, SEQ ID NO: 25 and SEQ ID NO: 27, and homologs thereof,and more particularly of a polypeptide which has the sequence SEQ ID NO:5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 16, SEQ ID NO: 25 or SEQ IDNO: 27, of its proteolytic fragments or of any synthetic peptide deducedfrom its sequence, for selecting novel antiviral molecules having fewerside effects.

A subject of the invention is also the use of a polypeptide, the peptidesequence of which is chosen from SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO:16, SEQ ID NO: 25 and SEQ ID NO: 27, and homologs thereof, and moreparticularly of a polypeptide which has the sequence SEQ ID NO: 5, SEQID NO 6, SEQ ID NO: 7, SEQ ID NO: 16, SEQ ID NO: 25 or SEQ ID NO: 27, ofits proteolytic fragments or of any synthetic peptide deduced from itssequence, for preparing specific antisera and/or monoclonal antibodiesaimed in particular at purifying said polypeptide and its fragments, orat modulating its activity.

By extension, a subject of the invention is also any use of saidsequence for producing recombinant antibodies or antibody fragments,whatever the biological system used to produce them.

A subject of the invention is also a polyclonal or monoclonal antibodycharacterized in that it specifically recognizes a polypeptide, thepeptide sequence of which is represented wholly or partly by SEQ ID NO:5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 16, SEQ ID NO: 25 or SEQ IDNO: 27, and more particularly which consists of the sequence SEQ ID NO:5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 16, SEQ ID NO: 25 or SEQ IDNO: 27.

The invention is also directed toward the use of this antibody, forpreparing a composition intended for the diagnosis of a deficiency in orof an overexpression of the SASPase protein (SEQ ID NO: 5).

It also relates to the use of an antibody, for preparing a compositionaimed at blocking the activity of and/or activating the SASPase in thetreatment of pathologies characterized by an overexpression and/or anexaggerated activity of the SASPase.

The antibody may be an antibody prepared by immunization of any animalspecies that can be used for this purpose, particularly the rabbit. Theantibody may be prepared by immunization using a polypeptide of theinvention, whether this polypeptide is of natural, synthetic orrecombinant origin, preferably purified.

It is known that a protein is synthesized in cells based on adeoxyribonucleic acid (DNA) matrix encoding said protein. It is alsoknown that the genetic code is degenerate. Thus, the amino acid sequenceof the polypeptide of the invention may be derived from variousdeoxyribonucleic acid sequences, which may be natural or synthetic. Theterm “synthetic deoxyribonucleic acid sequence” is here intended to meanany sequence obtained chemically or by genetic manipulation.

Said deoxyribonucleic acid sequences may be derived from any possibleorigins, namely either animal, in particular mammalian, and even moreparticularly human origin, or plant origin, or from microorganisms(viruses, phages, bacteria, inter alia) or else from fungi, withoutprejudice regarding whether or not they are naturally present in saidorganism of origin.

As it happens, the invention relates to the isolated and purifieddeoxyribonucleic acid fragments encoding the claimed polypeptides.

In the course of these studies, the applicant has been able to isolateand purify the deoxyribonucleic acid fragments encoding the primaryamino acid sequences of the polypeptides of sequence SEQ ID NO: 5, SEQID NO: 6, SEQ ID NO: 7, SEQ ID NO: 16, SEQ ID NO: 25 or SEQ ID NO: 27,from human skin.

A subject of the invention is an isolated and purified deoxyribonucleicacid fragment, the nucleotide sequence of which comprises at least thecoding nucleotide sequence SEQ ID NO: 24, and in particular isrepresented by the coding nucleotide sequence SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO: 24, SEQ ID NO: 26 or SEQ ID NO: 28.

The nucleic acid sequences according to the invention may in particularbe used for preparing corresponding sense or antisense ribonucleic acidsequences.

A subject of the invention is also any poly-nucleotide, ribonucleic acidor deoxyribonucleic acid, which may be sense or antisense, in particular“small interfering RNA”, corresponding at least to the coding nucleotidesequence SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 26 orSEQ ID NO: 28.

The nucleic acid sequences of the invention can also be used to prepareoligonucleotide primers which hybridize, under high stringencyconditions, with the sequence SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ IDNO: 24, SEQ ID NO: 26 and SEQ ID NO: 28.

These sense and/or antisense oligonucleotide primers may be useful forsequencing reactions or specific amplification reactions according tothe “PCR” (polymerase chain reaction) technique or any other variantthereof with the aim of cloning, identifying or diagnosing a polypeptiderepresented by SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO 5, SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 16, SEQ ID NO: 25or SEQ ID NO: 27.

In particular, the probes or primers of the invention are labeled priorto their use. For this, several techniques are within the scope of thoseskilled in the art, such as, for example, fluorescent, radioactive,chemiluminescent or enzymatic labeling.

The in vitro diagnostic methods in which these nucleotide probes areused for detecting synthesis of nucleic acid sequences encoding apolypeptide according to the invention are included in the presentinvention.

The DNA fragment corresponding to the sequence SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO: 24, SEQ ID NO: 26 or SEQ ID NO: 28 can also be introducedinto an expression vector, thus allowing the synthesis of acorresponding “recombinant” protein.

A subject of the invention is therefore also a recombinant expressionvector containing all or part of the coding nucleotide sequence SEQ IDNO: 19, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 26 or SEQ ID NO: 28.

A subject of the invention is also a cosmetic or pharmaceuticalcomposition comprising, in a physiologically acceptable medium, anatural or synthetic deoxyribonucleic acid sequence encoding the primaryamino acid sequence of a polypeptide in accordance with the invention ora sense or antisense ribonucleic acid sequence, in particularinterfering antisense ribonucleic acid, corresponding to said sequenceSEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 26 or SEQ ID NO:28.

In general, any composition of the invention may be ingested, injectedor applied to the skin (on any cutaneous zone of the body) or to themucous membranes (buccal, jugal, gingival, genital, conjunctival, etc.).

Preferably, a composition of the invention is applied to the skin or themucous membranes.

According to the method of administration considered, it may be in anyof the pharmaceutical forms normally used.

For topical application to the skin, the composition may have the formin particular of aqueous or oily solutions or of dispersions of thelotion or serum type, of emulsions with a liquid or semi-liquidconsistency of the milk type, obtained by dispersion of a fatty phase inan aqueous phase (O/W) or vice versa (W/O), or of suspensions oremulsions with a soft consistency of the aqueous or anhydrous cream orgel type, or else of microcapsules or microparticles, or of vesiculardispersions of the ionic and/or nonionic type or of foams. Thesecompositions are prepared according to the usual methods.

For injection, the composition may be in the form of aqueous or oilylotions or in the form of serums. For the eyes, it may be in the form ofdrops, and for ingestion, it may be in the form of capsules, ofgranules, of syrups or of tablets.

The amounts of the various constituents of the compositions according tothe invention are those conventionally used in the fields underconsideration.

In the cosmetics field, these compositions constitute in particularcleansing, protection, treatment or care creams for the face, for thehands, for the feet, for the large anatomical folds or for the body (forexample day creams, night creams, makeup-removing creams, foundationcreams, antisun creams), fluid foundations, makeup-removing milks,protective body milks or body care milks, antisun milks, skincarelotions, gels or foams, for instance cleansing lotions, antisun lotions,artificial tanning lotions, bath compositions, deodorant compositionscomprising a bactericidal agent, aftershave gels or lotions,hair-removing creams, insect-repellent compositions, pain-reliefcompositions, or compositions for treating certain skin diseases, suchas eczema, rosacea, psoriasis, lichens and severe pruritus.

The compositions according to the invention may also consist of solidpreparations constituting cleansing soaps or bars.

The compositions may also be packaged in the form of an aerosolcomposition also comprising a pressurized propellant.

A composition according to the invention may also be a care compositionfor the scalp, and in particular a shampoo, a setting lotion, a treatinglotion, a styling cream or gel, a dye composition (in particular foroxidation dyeing) optionally in the form of coloring shampoos, ofrestructuring lotions for the hair, a permanent-waving composition (inparticular a composition for the first stage of a permanent-wavingoperation), a lotion or a gel for preventing hair loss, an antiparasiticshampoo, an antidandruff shampoo, etc.

A composition may also be for orodental use, for example a toothpaste.In this case, the composition may contain adjuvants and additives thatare usual for compositions for oral use, and in particular surfactants,thickeners, wetting agents, polishing agents such as silica, variousactive ingredients such as fluorides, in particular sodium fluoride, andoptionally sweeteners such as sodium saccharinate.

When the composition is an emulsion, the proportion of the fatty phasemay range from approximately 5% to 80% by weight, and preferably fromapproximately 5% to 50% by weight, relative to the total weight of thecomposition. The oils, the waxes, the emulsifiers and the co-emulsifiersused in the composition in emulsion form are chosen from thoseconventionally used in the cosmetic field. The emulsifier and theco-emulsifier are present, in the composition, in a proportion rangingfrom 0.3% to 30% by weight, and preferably from 0.5% to 20% by weight,relative to the total weight of the composition. The emulsion may alsocontain lipid vesicles.

When the composition is an oily solution or gel, the fatty phase mayrepresent more than 90% of the total weight of the composition.

In a known manner, the cosmetic composition may also contain adjuvantsthat are common in the cosmetics field, such as hydrophilic orlipophilic gelling agents, hydrophilic or lipophilic additives,preserving agents, antioxidants, solvents, fragrances, fillers,screening agents, odor absorbers and dyestuffs. The amounts of thesevarious adjuvants are those conventionally used in the cosmetics field,and range, for example, from approximately 0.01% to 10% of the totalweight of the composition. Depending on their nature, these adjuvantsmay be introduced into the fatty phase, into the aqueous phase and/orinto the lipid spherules.

As oils or waxes which may be used in the invention, mention may be madeof mineral oils (liquid petroleum jelly), plant oils (liquid fraction ofkarite butter, or sunflower oil), animal oils (perhydro-squalene),synthetic oils (Purcellin oil), silicone oils or waxes (cyclomethicone)and fluoro oils (perfluoropolyethers), beeswax, carnauba wax or paraffinwax. Fatty alcohols and fatty acids (stearic acid) may be added to theseoils. As emulsifiers which may be used in the invention, mention may bemade, for example, of glyceryl stearate, polysorbate 60 and the mixtureof PEG-6/PEG-32/glycol stearate sold under the name Tefose® 63 by thecompany Gattefosse.

As solvents which may be used in the invention, mention may be made oflower alcohols, in particular ethanol and isopropanol, and propyleneglycol.

As hydrophilic gelling agents which may be used in the invention,mention may be made of carboxyvinyl polymers (carbomer®), acryliccopolymers such as acrylate/alkyl acrylate copolymers, polyacrylamides,polysaccharides such as hydroxypropylcellulose, natural gums and clays,and, as lipophilic gelling agents, mention may be made of modified clayssuch as bentones, metal salts of fatty acids, such as aluminumstearates, and hydrophobic silica, ethylcellulose and polyethylene.

The composition may contain other hydrophilic active agents, such asproteins or protein hydrolysates, amino acids, polyols, urea, allantoin,sugars and sugar derivatives, water-soluble vitamins, plant extracts andhydroxy acids.

Lipophilic active agents which may be used include retinol (vitamin A)and derivatives thereof, tocopherol (vitamin E) and derivatives thereof,essential fatty acids, ceramides, essential oils, and salicylic acid andderivatives thereof.

According to the invention, the composition may combine at least oneother active agent intended in particular for the prevention and/ortreatment of skin ailments. Among these active agents, mention may bemade, by way of example, of:

-   -   agents for decreasing differentiation and/or proliferation        and/or pigmentation of the skin, such as retinoic acid and        isomers thereof, retinol and esters thereof, vitamin D and        derivatives thereof, estrogens such as estradiol, kojic acid or        hydroquinone;    -   antibacterial agents, such as clindamycin phosphate or        erythromycin or antibiotics of the tetracycline class;    -   antiparasitic agents, in particular metroni-dazole, crotamiton        or pyrethrinoids;    -   antifungal agents, in particular compounds belonging to the        imidazole class, such as econazole, ketoconazole or miconazole,        or salts thereof, polyene compounds, such as amphotericin B,        compounds of the allylamine family, such as terbinafin, or        alternatively octopirox;    -   antiviral agents such as acyclovir;    -   steroidal anti-inflammatory agents, such as hydrocortisone,        betamethasone valerate or clobetasol propionate, or nonsteroidal        anti-inflammatory agents such as, for example, ibuprofen and        salts thereof, diclofenac and salts thereof, acetylsalicylic        acid, acetaminophen or glycyrrhizic acid;    -   anesthetics, such as lidocaine hydrochloride and derivatives        thereof;    -   antipruriginous agents, such as thenaldine, trimeprazine or        cyproheptadine;    -   keratolytic agents, such as α- and β-hydroxy-carboxylic acids or        β-keto carboxylic acids, their salts, amides or esters and more        particularly hydroxy acids such as glycolic acid, lactic acid,        salicylic acid, citric acid and fruit acids in general, and        5-n-octanoylsalicylic acid;    -   free-radical scavengers, such as a-tocopherol or esters thereof,        superoxide dismutases, certain metal chelating agents, or        ascorbic acid and esters thereof;    -   antiseborrheic agents, such as progesterone;    -   antidandruff agents, such as octopirox or zinc pyrithione;    -   antiacne agents, such as retinoic acid or benzoyl peroxide.

Thus, according to the particular embodiment, the composition accordingto the invention also comprises at least one agent chosen fromantibacterial, antiparasitic, antifungal, antiviral, anti-inflammatoryand antipruriginous agents, anesthetics, keratolytic agents,free-radical scavengers, antiseborrheic agents, antidandruff agents,antiacne agents and/or agents for decreasing differentiation and/orproliferation and/or pigmentation of the skin.

The examples that appear hereinafter are presented by way of nonlimitingillustration of the invention.

FIGURES

FIG. 1: photograph of the 2D electrophoresis gel obtained according toexample I,

FIG. 2: representation of the influence of pH on the proteolyticactivity of the SASPase,

FIG. 3: analysis of the activity of a certain number of conventionalmodulators with respect to the proteolytic activity of the SASPase,

FIG. 4: representation of the structural homologies between the SASPase(SEQ ID NO: 39) and proteases FIAV (SEQ ID NO: 37) and HIV2 (SEQ ID NO:38),

FIG. 5: representation of the hydrolysis of the substrates representedby the sequences SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO:34 and SEQ ID NO: 35, by the SASPase.

EXAMPLES

Materials

The oligonucleotides used in the various experiments are given below intable I.

TABLE I Primer SEQ ID name Sequence 5′ to 3′ N° SC 130GATAGGATCCATGGCCGGGAGCGGAGCCAGGAG 12 SC 131TTGAATTCTCAGTGGGATAGCTCCTGCCGC 11 SC 134 GGCCCTGGGTGTCTACAATA 13 SC 135TTGGCCACCTTTACCACATT 14 SC 140 TAGGATCCATGGGGAGCCCAGGGGC 10 Not I-(dT)₁₈is an oligonucleotide sold by the company Amersham Pharmacia Biotech NotI-(dT)₁₈ is designed so as to bind to long series of A, for example polyA+ series. Used in reverse transcription reactions, Not I-(dT)₁₈ bindsto the poly A+.

Example I Identification and Isolation of the SASPase by Two-DimensionalGel Electrophoresis and Sequencing

a) Preparation from Human Epidermis

Buffers:

I: 0.3% SDS; 28 mM tris-HCl; 22 mM tris-base

2D: 8M urea; 2% (w/v) 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS); 20 mM dithiothreitol; 0.5% (v/v) buffer Imobiline pHgradient (IPG), pH=3 9-10, 18 cm, sold by the company Amersham PharmaciaBiotech.

Extracts of reconstructed human epidermis are prepared from 30 units ofthe D13 Episkin kit. 15 ml of buffer I are added to the 30 reconstructedepidermides and the entire mixture is homogenized with a Potterhomogenizer, brought to the boil for 10 min, and then again homogenizedwith a Potter homogenizer. The solution is then centrifuged at 10 000 gfor 10 min. The supernatant is collected and filtered through a 0.22 μmmembrane. 12 ml of supernatant SI are thus obtained. Cold acetone (10v/2 v) is then added to the supernatant SI. After incubation for 20 min,the mixture obtained is centrifuged at 9400 g for 10 min. Thesupernatant is then removed and the pellet is dried at ambienttemperature for 20 min. The pellet is then taken up in 2 ml of 2Dbuffer. The extract EI is thus obtained. The final protein concentrationis 11 mg/ml.

b) Two-Dimensional Gel

The two-dimensional separation of the proteins contained in the extractEI is carried out on a Pharmacia device (model Multiphor II). Thetwo-dimensional separation of the proteins was carried out according tothe supplier's recommendations, except that, for the re-equilibration ofthe IPG gel after migration in the first direction, the iodoacetamidewas omitted. The staining of the spots, the recovery thereof and thesequencing of the polypeptides that they contained were carried outaccording to the techniques described in Mehul B, Bernard D, SimonettiL, Bernard M A, Schmidt R: Identification and cloning of a newcalmodulin-like protein from human epidermis. J Biol Chem 275:12841-12347, 2000, or else “A practical guide to protein and peptidepurification for microsequencing” (editor Paul Matsudaira, secondedition, 1993). Represented in FIG. 1 is the correspondingelectrophoresis gel.

The proteins were detected by amido black staining. The spotscorresponding to proteins identified by Edman sequencing are locatedwith the name of these proteins. The spot called SASPase makes itpossible to locate an epidermal form of the protein having an apparentMW of 12 kD and a pI of 5.8.

The results obtained made it possible to characterize the sequences SEQID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.

Example II Isolation of the cDNA Encoding the SASPase from HumanKeratinocytes and Expression of the SASPase (SEQ ID NO: 5) and of itsTruncated (Δ1-84) Form (SEQ ID NO: 4)

a) Preparation of the cDNA

The total RNA of keratinocytes originating from reconstructed humanepidermis after 13 days of culture was prepared using the “RNeasy kit®”for preparing RNA, and purified using the “QIAshredder column®” kit,sold by the company Qiagen, according to the supplier's instructions.

The complementary DNAs (cDNAs) of the RNAs thus prepared weresynthesized using the “First Strand cDNA Synthesis®” kit sold by thecompany Amersham Pharmacia Biotech, according to the supplier'sinstructions, using the oligonucleotide Not I-(dT)₁₈ as primer.

cDNA fragments encoding the complete SASPase thus obtained wereamplified by polymerase chain reactions (PCRs) in a “Thermocycler®”device sold by the company Perkin-Elmer, using a DNA polymerase, pfu,sold by the company Promega and, as primers, the pair ofoligonucleotides SC140 (SEQ ID NO: 10)/SC131 (SEQ ID NO: 11) and thefollowing conditions: 1 cycle (95° C. for 2 min), 35 cycles (94° C. for30 sec, 65° C. for 30 sec, 72° C. for 2 min) and 1 cycle (72° C. for 7min).

Similarly, cDNA fragments encoding the truncated form of the SASPase(SEQ ID NO: 4), lacking the 84 N-terminal amino acids of the SASPase,referred to as Δ1-84, were amplified by PCR using, as primers, the pairof oligonucleotides SC131/SC130 (SEQ ID NO: 11 and SEQ ID NO: 12).

b) Construction and Expression of the Recombinant SASPase (rSASPase)

The SASPase cDNA obtained above is introduced into the plasmid vectorpGex-4T-3 sold by the company Amersham Pharmacia Biotech, bycleavage/ligation at the BamH-1/EcoR1 restriction site. This recombinantplasmid which contains, in phase in the reading frame, the codingsequence of the SASPase (SEQ ID NO: 5) and the coding sequence ofglutathione S-transferase (GST) is then introduced into E. coli strainBL21 (DE3) sold by the company Amersham Pharmacia Biotech. Therecombinant fusion protein expressed by the bacteria can be cleaved withthrombin under gentle conditions, the construct being such that thefusion protein obtained carries a site for cleavage by this protease.

The expression product is purified by affinity chromatography on agluthatione-sepharose column.

All these experiments were carried out by strictly applying the variousprotocols from the suppliers.

The SDS-PAGE gel electrophoresis analysis of an aliquot fraction of theexpression product obtained by carrying out the method described aboveshows that this method makes it possible to obtain a satisfactory amountof the recombinant fusion protein GST-SASPase.

Similarly, the expression, in a satisfactory amount, of the recombinantfusion protein GST-truncated SASPase Δ1-84 (SEQ ID NO: 4) was obtained.

Example III Obtaining the Activated SASPase (SEQ ID NO: 6)

The recombinant fusion protein GST-SASPase Δ1-84 (SEQ ID NO: 4) elutedfrom the glutathione-sepharose column, obtained in example II, isincubated with thrombin in PBS buffer, at pH 8, for 18 hours at 22° C.The buffer is exchanged by filtration on Biorad G25® gel against a 100mM acetate solution, pH 4.5. The mixture thus obtained is subjected tocationic chromatography using an NaCl gradient (0 to 1 M NaCl). Thefractions containing the GST and the thrombin are eliminated and thefraction eluted with the 750 mM NaCl solution is recovered. An SDS-PAGEgel electrophoresis analysis carried out on the fraction eluted with the750 mM NaCl solution, containing caseinolytic activity (data not shown),shows the presence of a major band which, by comparison with themolecular mass markers, has an apparent molecular mass of 12 kD and aminor band which corresponds to the truncated SASPase form Δ1-84 (SEQ IDNO: 4).

The 12 kD form corresponds to the activated form of the SASPase. TheEdman sequencing indicates that the isolated product corresponds to theactivated SASPase (SEQ ID NO: 6).

Example IV Obtaining the Activated SASPase (SEQ ID NO: 25)

The deletion of the site encoding the FANS sequence on the truncatedform of the SASPase (SEQ ID NO: 4), using the Quick Change Site-directedMutagenesis kit (Stratagene) and suitable primers, is carried out usingas DNA matrix the plasmid construct of the SASPase C27 sequence (SEQ IDNO: 4) in the vector pGEX-4T3.

A protein, the protein sequence of which is represented by SEQ ID NO:36, is obtained.

After incubation of this new protein at pH 5.00 in acetate buffer, thereis, surprisingly, generation of a low molecular weight fragment(approximately 21 kDa by SDS-PAGE electrophoresis) which has at most thesequence SEQ ID NO: 25 of theoretical mass 18731.44.

SEQ ID NO: 27, of theoretical mass 16794.43, is obtained from SEQ ID NO:25 by assuming a C-terminal activation identical to the form SEQ ID NO:6.

Example V Characterization of the Proteolytic Activity of the SASPaseProtein

a) Activity with Respect to a Substrate

This activity was demonstrated according to the following protocol:

The oxidized beta-chain of insulin (Sigma) is used as substrate. Theconcentration is 50 μg in 1.5 ml of 0.1 M acetate buffer, pH 5.0. 50 μlof 12 kD SASPase (SEQ ID NO: 6) (approximately 1 mg/ml) purified by gelfiltration are added so as to initialize the hydrolysis. Controlswithout enzyme or without substrate are used. Over time (1 h to 24 h at37° C.), HPLCs are performed in order to follow the hydrolysis. Thepeaks that appear rapidly correspond to the main hydrolysis sites andthose that only appear after 24 h correspond to the secondary sites. Thepeaks are collected after they have been fractionated, and areN-terminal sequences (EDMAN sequencing, Pasteur Institute).

FVNQHLCGSHLVEALYLVCGERGFF. (SEQ ID NO: 15)

Main site: E/A (pepsin-like)

Secondary sites: L/Y and Y/L (pepsin-like)

These results show that the activated SASPase is capable of degradinginsulin. Heat denaturation (95° C., 10 min) of the SASPase abolishes itsinsulin-degrading activity. Its caseinolytic activity was, moreover,demonstrated (results not shown).

b) Autocatalytic Activity

In this second assay, it is the GST-SASPase protein (SEQ ID NO: 4) whichis itself used as a substrate by autocatalysis.

The GST-SASPase, at 3 mg/ml in 0.1 M phosphate buffer, pH 7.0,containing 50% glycerol, is rapidly acidified to pH 5.0 with 1 M acetatebuffer, pH 4.5. At each incubation time, at 37° C., an aliquot is takenand the reaction is blocked by adding an equivalent volume of Laemmlibuffer without DTT. At the end of the kinetics, each sample is analyzedby SDS-PAGE electrophoresis (15% acrylamide gel), demonstrating agradual appearance of a major band that migrates at an apparent MW of 12kD subsequent to the disappearance of the fusion protein.

This assay shows that it is also possible to obtain the activatedSASPase (SEQ ID NO: 6) directly by acidification at pH 3 to 6,preferably 4 to 6, of the recombinant fusion protein GST-rSASPase Δ1-84or GST-SASPase obtained in example I, followed by a gel filtration (G75)purification step.

Furthermore, analysis of the activated solutions by Edman sequencing andby QTOF mass spectrometry gives, for the autoactivated protease, threecleavage sites that are more or less equivalent in terms ofprobabilities:

F/A (like certain metalloproteases in the skin)

N/S (cleavage site not listed)

E/L (only described for matrilysin, which is capable of activatingurokinase and MMPs 1, 2 and 9).

Example VI Site-Directed Mutations on the Active Site of the SASPaseProving that it Belongs to the Aspartic Acid Protease Family

The Quick Change Site-directed Mutagenesis kit (Stratagene) is used forproducing the SASPase mutants.

The SASPase sequence SEQ ID NO: 5 is modified at aspartic acid No. 212,i.e. on its potential active site. To do this, two oligonucleotides aresynthesized such that amino acid 212 is substituted either to alanine(mutant A/D), or to glutamic acid (mutant E/D).

A PCR amplification is carried out with each pair of mutatedoligonucleotides, and the plasmid construct of the SASPase C27 sequencein the vector pGEX-4T3 is used as DNA matrix.

Competent bacteria (XL1 blue) are then transformed with each PCRproduct. Several clones are amplified and sequenced in order to verifythe presence of the desired mutation and only of this mutation.

Autocleavage of the SASPase C27 and of its Mutants:

The mutated recombinant proteins are produced according to the sameprotocol as for the wild-type form, SASPase C27; (SEQ ID NO: 4).

Each recombinant protein produced in fusion protein form is thenincubated in a 1 M acetate buffer, pH 4.5, at 37° C. Samples are takenover time and are analyzed by SDS-PAGE.

A decrease in the whole form GST-SASPase C27 is observed over time,which demonstrates an autocleavage and, consequently, an autoactivation;numerous lower molecular weight bands appear. The mutated forms (A/D)and (E/D), for their part, do not autoactivate.

This experiment confirms the involvement of aspartic acid No. 212 in theactive site of the SASPase.

Example VII Analysis of the SASPase Expression by Northern Blotting,RT-PCR in Human Tissues and in Keratinocytes

The analyses were carried out by Northern blotting, using commercialpolyA+RNA blot membranes (Ambion®) according to the protocol describedby the manufacturer, and by RT-PCR using a cDNA collection of the human“rapid-scan®” membranes sold by the company OriGene Technologies, Inc.

The Northern blotting analysis was carried out using, as probe, the cDNAencoding the (truncated Δ1-84) form of the SASPase (SEQ ID NO: 4)isolated from the recombinant plasmid prepared in example II, and usinga membrane containing various RNA samples.

The PCR is carried out using the Taq polymerase sold by the companyPromega, and the pair of oligonucleotides SC134 (SEQ ID NO: 13)/SC135(SEQ ID NO 14) as primers, under the following conditions:

-   -   1 cycle of 3 min at 94° C.,    -   25 to 30 cycles (94° C. for 30 sec, 53° C. for 30 sec, 72° C.        for 60 sec), and    -   1 cycle at 72° C. for 5 min.

The resulting PCR products are visualized by ethidium bromide stainingafter separation on a 2% (weight/volume) agarose gel.

From the results, it emerges that the SASPase is substantially expressedin virtually all the tissues tested, at a low level in fetal liver,fetal brain, ovary, adrenal gland, thyroid, placenta, testes, stomach,muscle, lung, liver, spleen and heart, and at an even lower level inbone marrow, pancreas, saliva and small intestine. This expression is,on the other hand significant in the brain and particularly very high inthe skin.

Example VIII Oligomerization of the SASPase Using a Crosslinking Reagent

The protocol used is based on the technique described in T. D. Meek etal.; Proc, Natl. Acad. Sci. USA vol. 86, pages 1841-1845, March 1989.

A study of the crosslinking using BS3 (Pierce) is carried out onactivated SASPase (SEQ ID NO: 6), obtained by acidification andpurification by exclusion chromatography (gel filtration) of therecombinant fusion protein GST-SASPase Δ1-84.

1 μg of BS3 in 60 μl of 50 mM phosphate buffer, pH 7, containing 150 mMNaCl, 0.1% TX100 and 5 mM EDTA is added to the activated SASPase (SEQ IDNO: 6) obtained in example III, placed in ice. A control sample free ofBS3 is also prepared. After 90 min, 2 μl of 1 M Tris-HCl, pH 8, areintroduced into the reaction media in order to stop the reaction. Theproducts obtained are then analyzed by gel filtration and by SDS-PAGEelectrophoresis.

The activated SASPase incubated with BS3 forms a stable multimericcomplex which, by gel filtration, separates into three major peaks.Three distinct bands can also be observed on a gel after SDS-PAGEelectrophoresis, the apparent molecular masses of which bands,determined by comparison with the molecular mass markers, arerespectively 12 kD, and between 10-14 kD, 30-45 kD and 60-100 kD.

Example IX Influence of pH on the Proteolytic Activity of SASPase

10 μl of GST-SASPase Δ1-84 fusion proteins (approximately 3 mg/ml)obtained in example II are incubated in 200 μl of 0.1 M acetate buffer,adjusted to various pH values in the presence of the casein substratesold under the name Enzchek® by the company Molecular Probes, and usedat the concentrations recommended by the supplier.

Each assay is repeated three times.

After incubation at 37° C. for 20 hours, the fluorescence is measured ona plate reader sold under the name Biolumin® by the company MolecularProbes, using the excitation/emission couple: 485/535 nm. The resultsare given in FIG. 2.

These results show that, under the experimental conditions selected, theenzymatic activity of the SASPase is pH-dependent and is optimal at pH5. Moreover, assays that are not shown, in 0.1 M phosphate buffer of pH6 to 7.5, show only a weak residual activity.

The influence of pH on the autoactivation is also determined and showsthat the optimum pH is between 3 and 6.9, and preferably between 4 and6.

Example X Study of the Effect of Various Inhibitors of the Aspartic AcidProtease Family on the Caseinolytic Activity of the Activated SASPase(SEQ ID NO: 6)

10 μl of activated SASPase (SEQ ID NO: 6) that have been autoactivated(approximately 3 mg/ml) are added to 200 μl of 0.1 M acetate buffer, pH5.5, containing 20 μl of DMSO with retropepsin inhibitors. A controlassay is carried out under the same conditions in the absence ofinhibitor.

The mixture is pre-incubated at 4° C. for 1 hour, and the substrate,casein, sold under the name Enzchek® by the company Molecular Probes, isthen added in sufficient amount to obtain the concentration recommendedby the supplier, the solutions having been heated to 37° C. beforehand.

The casein hydrolysis is followed by measuring the fluorescence on aBiolumin® plate reader sold by the company Molecular Probes, using theexcitation/emission couple: 485/535 nm.

The results obtained are given in FIG. 3. It is noted that the kineticsfor casein hydrolysis, in the presence of the RP1 and RP2 retropepsininhibitors, are lower than the control. The activity of the SASPase istherefore inhibited by these inhibitors.

-   -   RP1 corresponds to the sequence Ac-Leu-Val-Phe-aldehyde and is        sold by Bachem under the reference N 1395.0005,    -   RP2 corresponds to the sequence Ac-Leu-Leu-Met-aldehyde and is        sold by Bachem under the reference N 1315.0005 and    -   RP3 corresponds to the sequence Ac-Leu-Leu-Nle-aldehyde and is        sold by Bachem under the reference N 1320.0005.

It is also noted that the kinetics for casein hydrolysis, in thepresence of the RP3 retropepsin inhibitor, are significantly higher thanthose obtained under the control conditions. The activity of the SASPasetherefore appears to be stimulated by this inhibitor.

Example XI Study of the Effect of the Activated SASPase (SEQ ID NO: 6)on the Degradation of Corneodesmosin Extracted from Human StratumCorneum

a) Principle

Corneodesmosin is a marker for desquamation since it is involved incorneocyte cohesion at corneodesmosomes. The more the protein isdegraded, the more significant is the detachment of the corneocytes.Corneodesmosin degradation is therefore a key step of desquamation.

The test used in this study therefore consists in following thisdegradation in the presence of the test substance.

b) Preparation of the Sample

Acetone powders are prepared using stripping varnish (Mehul B, BernardD, Simonetti L, Bernard M A, Schmidt R: Identification and cloning of anew calmodulin-like protein from human epidermis. J Biol Chem 275:12841-12347, 2000), taken from the lower parts of the legs of volunteerswith dry skin. Aliquot fractions of 2 mg of stratum corneum powder areintroduced separately into Eppendorf tubes, and then immersed in thetest solutions in a proportion of 100 μl/mg. The solutions are preparedin acetate buffer, pH 5. A control without protease is prepared inparallel in order to evaluate the natural degradation of corneodesmosin.For each test, three samples are prepared. The samples, trials andcontrol, are incubated at 30° C., with stirring for 24 hours.

c) Extraction, Separation and Detection of Proteins

The proteins are extracted with complete Laemmli buffer. They areassayed by the Bradford method (Bio-Rad® kit). The concentration of eachsample is adjusted so as to allow comparison of the samples. Thepolypeptides are separated by SDS-PAGE electrophoresis on a 15%acrylamide gel, and then transferred onto a PVDF membrane.Immunodetection with a solution of anti-corneodesmosin antibody used at1/12 500 and of secondary antibody coupled to peroxidase makes itpossible to visualize the corneodesmosin. The bands detected bychemiluminescence are quantified using the Quantity One® program fromthe company Biorad. The membranes are then stained with amido-black, andthen scanned. In addition, the keratins, which are major proteins ofcorneocyte extracts, are quantified in order to verify the adjustment to0.6 mg/ml of all the samples.

A positive control for degradation (+) is prepared using 5 mM of EDTA. Atrial is carried out in the presence of 60 μg of activated SASPase. Anegative control represents the natural degradation of corneodesmosinunder the operating conditions of the test.

d) Results

TABLE II Standardized % Residual corneodesmosin corneodesmosin T0 T24 hT24 h/T0*100 Activated SASPase 33107 24918 75 +control 31657 26685 84−control 32701 30546 93

A significant decrease in the percentage of residual corneodesmosin isobserved when the stratum corneum acetone powders are brought intocontact with the activated SASPase (SEQ ID NO: 6), compared with thecontrols.

Example XII Investigation of Substrate for Demonstrating the SASPaseActivity

The peptide substrate considered (SEQ ID NO 31, SEQ ID NO: 32, SEQ IDNO: 33, SEQ ID NO: 34 or SEQ ID NO: 35) quenched using the Abz/(NO₂)Tyrfluorescent system is used at 100 μM in DMSO, and then 10 μl of thisstock substrate solution are added per 100 μl of 0.1 M acetate buffer,pH 5.00.

10 μg of GST-C27 (fusion protein at 1 mg/ml in PBS) are added.Incubation is carried out at 37° C. The fluorescence is read at 340/450nm on a Biolumin plate reader at various times. The results are given inFIG. 5.

It is noted that the natural (wt) peptide is effectively hydrolyzed bythe SASPase, but amino acid substitutions in the sequence of thispeptide can generate substrates for which the SASPase has greateraffinity.

A search (MEROPS base) of the various proteases capable of cleavingpeptide bonds that are hydrolyzed or hydrolyzable by the SASPase(combinations of the various amino acids found in position P1 with theamino acids found in position P2 of the sequences hydrolyzed by theSASPase) reveals, in parallel with retroviral-type proteases, proteaseswhich have been described as being important for skin: SCCE(desquamation, conversion of proIL1B), MMP1, MMP2 and MMP9(cicatrization, etc.), mu Calpain (maturation of the cornified envelope,filaggrin processing), thrombin (activation of protease activatedreceptors), convertases 1, 2, 4, 5 and 7 (filaggrin processing,regulation of differentiation, etc.).

These observations support the idea that the SASPase has an importantrole in skin physiology.

1. A cosmetic or pharmaceutical composition comprising, in aphysiologically acceptable medium, at least one purified, natural orsynthetic polypeptide, wherein said polypeptide consists of at least oneamino acid sequence selected from the group consisting of SEQ ID NO: 4,SEQ ID NO: 5, and SEQ ID NO:
 6. 2. The composition as claimed in claim1, wherein said polypeptide consists of SEQ ID NO: 5 or SEQ ID NO:
 6. 3.The composition as claimed in claim 1, wherein said polypeptide isfurther prepared as a dimeric or other multimeric form.
 4. Thecomposition as claimed in claim 1, wherein said polypeptide has furtherundergone one or more post-translational modifications.
 5. Thecomposition as claimed in claim 2, wherein said polypeptide is furtherfused with another polypeptide, a hydrophilic or hydrophobic targetingagent or a bioconversion precursor.
 6. The composition as claimed inclaim 1, wherein said polypeptide consists of SEQ ID NO:
 6. 7. Anisolated and purified polypeptide belonging to the aspartic acidprotease family and consisting of SEQ ID NO:
 6. 8. The polypeptide asclaimed in claim 7, having an apparent molecular mass of between 5 and30 kD.
 9. The polypeptide as claimed in claim 8, having an apparentmolecular mass of between 9 and 15 kD.
 10. The polypeptide as claimed inclaim 9, having an apparent molecular mass of between 11 and 14 kD. 11.The polypeptide as claimed in claim 7, wherein said polypeptide isfurther prepared as a dimeric or other multimeric form.
 12. Thepolypeptide as claimed in claim 7, having a theoretical isoelectricpoint of between 3 and
 9. 13. The polypeptide as claimed in claim 7,said polypeptide being of natural origin and purified from mammaliantissues.
 14. The polypeptide as claimed in claim 13, wherein saidpolypeptide is purified from human epidermis or other human skin. 15.The polypeptide as claimed in claim 7, wherein said polypeptide hasfurther undergone one or more post-translational modifications.
 16. Thepolypeptide as claimed in claim 7 wherein said polypeptide is furtherfused with another polypeptide, a hydrophilic or hydrophobic targetingagent or a bioconversion precursor.
 17. A method for degradingcorneodesmosin in corneocytes comprising applying to corneocytes aneffective corneodesmosin degrading amount of at least one polypeptide,wherein said polypeptide comprises at least one amino acid sequenceselected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5 and SEQID NO: 6.